The mechanism of excitation-contraction coupling

This chapter is most relevant to Section L1(iii) from the 2017 CICM Primary Syllabus, which expects the exam candidates to "describe the mechanism of excitation-contraction coupling". This has appeared at least a couple of times, once for each muscle tissue type (Question 8 from the first paper of 2014 for skeletal muscle, and 

Question 18 from the first paper of 2012 for smooth muscle). Both listed SAQs had dismal pass rates (34% and 20% respectively), suggesting the trainees were caught off-guard. Thus, a short answer-like summary is presented here, for future candidates to store in their synaptic granules.

To simply throw the string "excitation-contraction coupling" into a market-dominant search engine should usually yield a vast plethora of peer-reviewed and homecooked literature on this subject. The real skill of the reviewer is revealed in the sort of recommendations they can make after filtering through these. A candidate with limited time resources will obviously go for the single shortest simplest explanation, such as this review by Sweeney & Hammers (2018).  Another excellent resource is this thing from columbia.edu: it appears to be a scanned copy of a photocopied textbook chapter, distributed as a part of some course in 2004 and exposed for the world to see by some wonderfully porous folder permission settings, presumably dating back to Web 1.0. In-depth professional works encompassing the entire topic are not available, as nobody seems to have the stamina to cover all three tissue types in detail, and the reader needs three separate papers.  Calderón et al (2014) is great for skeletal muscle, Stern (1992) for cardiac and Somlyo (1985) or Endo (1983) for smooth muscle (yes, these references are forty years old, but they are free and the for the casual reader the main points remain accurate).

Definition of excitation-contraction coupling

Where do the boundaries of this topic lay? Most unready people, confronted with the question, would still be able to blurt out a passable definition, because the wording of the term is fairly unambiguous. It was coined in 1952 by Alexander Sandow, who used it to designate "the entire sequence of reactions - excitation, inward acting link, and activation of contraction". The nomenclature was clearly successful, insofar as the success of any terminology can be measured by its longevity. Seventy years on, we still have people referring to it by the same words, and as Dirksen et al (2022) have pointed out, the use of the word "coupling" has metastasised into other areas of medicine and biology, such that we see numerous "like-named franchises for secretion, transcription, and other couplings". 

The modern reader, looking for a  straight answer, would probably not find one better than the definition offered by Smith et al (in UpToDate): 

"the series of events that link the action potential (excitation) of the muscle cell membrane (the sarcolemma) to muscular contraction. "

This means the most important points to cover in a written exam answer are:

• Action potential propagation along the sarcolemma (including the propagation of this potential down into penetrating T tubules, when one is discussing cardiac or skeletal muscle)
• Calcium entry into the myocyte
• Calcium-mediated release of calcium from the sarcoplasmic reticulum
• ATP dependent contraction process, where CICM examiners apparently expected a "brief outline of the structure of a sarcomere and how it facilitates shortening" and "knowledge of the ATP dependent walk along processes of myosin heads"
• Activation of the sarcoplasmic calcium buffering system
• Resequestration of calcium into the sarcoplasmic reticulum, which is the mechanism of return to the relaxed state.

Additionally, predicting a relentless march towards increasingly more esoteric exam papers, the candidates should prepare to answer questions that ask them to compare the excitation-contraction coupling between the different types of muscle, and this is attempted at the very end. 

Action potential propagation along the sarcolemma

We could pick up this thread from the generation of the endplate potential by the opening of the nicotinic receptors at the neuromuscular junction, which would be a classical point of reference to start discussing the action potential of muscle cells, but this would be unfairly biased in favour of skeletal muscle. It is probably worth noting that the other muscle tissues have substantially different excitable behaviour. The best reference for this, unfortunately, was a group of chapters from the excellent Cell Physiology Source Book by Nicholas Sperelakis (1995), spanning about 100 pages; and no sane person would ever recommend these as essential exam preparation material. To spare the reader, the main differences can be summarised in a table:

	Skeletal muscle	Cardiac muscle	Smooth muscle
Electrical union	Each cell is electrically isolated	Connected by intercalated discs ("electrical syncytium")	Variably connected by gap junctions, depending on the tissue
Neural connection	Each cell has its own motor endplate	Most cells are not directly innervated	Most cells are not directly innervated
Resting membrane potential	-80 mV	-90 mV	From -50 to -70 mV
AP duration	4 ms	200-300 ms	Seconds, minutes...
Conduction velocity	2-5 m/s	0.4-0.9 m/s	15-1.6 cm/s

Skeletal muscle action potentials are ridiculously quick. The typical skeletal myocyte action potential "spike" lasts about four milliseconds, which is only slightly slower than the slowest of neurons. Here's a representative recording of a frog semitendinosus fibre from Sperelakis et al (1973), demonstrating all the important features (as well as some recording artifact):

Cardiac myocytes have a much longer action potential, lasting 200-300 milliseconds. There is already enough written about it in the cardiovascular physiology section, but here's a diagram anyway, in the unlikely case that the reader is somehow unable to recall this very characteristic shape:

This is about a hundred times slower than skeletal muscle, but still relatively quick compared to smooth muscle cells, which can remain depolarised for whole minutes. For example, here is a recording of the transmembrane potential from some vascular smooth muscle being tortured with noradrenaline by Hermsmeyer (1982):

As you can see, this vascular smooth muscle remained depolarised for the duration of the stimulus, and this weird behaviour is typical for vascular smooth muscle cells. In fact there is a lot of weirdness in smooth muscle electrical behaviour:

• The action potential magnitude and duration is extremely variable. As smooth muscle cells are variably innervated and receive a lot of their stimulus from paracrine and endocrine mediators, they are also very flexible in the duration and magnitude of their action potentials, even within the same tissue type. This massive variability in action potentials can be contrasted to the fairly uniform and predictable responses of skeletal and cardiac muscle, which always do the same basic thing when stimulated.
• Electrical connectivity is variable. Smooth myocytes are sometimes connected by gap junctions, but don't necessarily have to be. Not all smooth muscle tissues need to be connected electrically. For example "multi-unit" smooth muscle tissue functions as an anarchist syndicate of vaguely cooperative but otherwise independent cells that might each receive their own innervation. 
• Automaticity is normal. Normal smooth muscle cells are occasionally capable of spontaneous electrical activity, whereas skeletal muscle and most normal cardiac myocytes are not.
• Excitation does not have to be coupled to contraction.  Contraction can occur without electrical excitation, for example where the binding of a neurotransmitter hormone leads to the intracellular release of calcium from the sarcoplasmic reticulum without any need for an action potential or exogenous calcium.

After all this it will not surprise the reader to learn that the rate at which the action potential propagates along a myocyte will also differ depending on which types of muscle cells are involved. Intact skeletal muscle fibres enjoy rapid conduction, with a minimum action potential velocity similar to that of unmyelinated nerves -  something like 2-5 metres per second, according to amphibian muscle experiments by Sheikh et al (2001) and human data by Troni et al (1983).  Just as with nerves, the width of a fibre seems to matter - i.e. thicker fibres conduct faster. Cardiac myocytes conduct more slowly, at a rate of perhaps 0.4 to 0.9 m/s, which is fine because the heart is a rather compact organ (compared to, for example, the 60cm long sartorius) and the action potential does not have far to travel. Predictably, the slowest of all are smooth muscle sheets, with conduction velocities ranging from the relatively brisk 15 cm/sec for the human oesophagus to the embarrassingly sluggish 1.6 cm/sec for the retractor muscle of a dog's penis (Burnstock & Prosser, 1960). 

The propagation of the action potential into T tubules

For the thick meaty fibres of skeletal and cardiac muscle to contract rapidly in a coordinated fashion, the action potential needs to propagate deep into the interior of the cell, and it does so by means of the T tubules. Hill (1949) famously detected that it takes less than 40 milliseconds for excitation to make its way into the centre of the cell (in his case, a cardiac myocyte with a radius of 50 µm), and this phenomenon remained unexplained until the T tubules were discovered during the explosion of electron microscopy in the mid-1950s. As their importance is tightly linked to the importance of calcium, and the importance of calcium is the topic of the next section, not much more needs to be said about these structures, other perhaps than the statement that they tend to be more abundant where calcium cycling is more rapid. The mouse, whose resting heart rate is 500 beats per minute, has a higher density of T tubules in its cardiac myocytes than slower-paced animals, perhaps to facilitate a faster ion exchange by harnessing a larger surface area (Heinzel et al, 2002).  By extension of the same principle, smooth muscle has no need for T tubules, as the smooth muscle cells are relatively small and nothing involving smooth muscle needs to happen especially quickly. 

Calcium entry into muscle cells

One way or another, intracellular calcium is central to muscle function because it is a necessary cofactor for the contractile apparatus, and it can be made available to those proteins in two ways: it can be acquired from the extracellular fluid, or it can be released from the sarcoplasmic reticulum. Most muscle tissue types tend to do a bit of both. Cardiac myocytes tend to rely on a supply of extracellular calcium for their function, whereas skeletal and smooth muscle tend to rely more on the intracellular calcium already stored in their sarcoplasmic reticulum. As usual, there is no single paper that compares these mechanisms, but a good reference for each muscle tissue type is supplied below. 

• Skeletal muscle cells have L-type voltage-gated calcium channels on the surface membrane of their T tubules, usually referred to as dihydropyridine receptors or dihydropyridine channels because 1,4-dihydropyridine tends to block them. No, currently available dihydropyridine calcium channel blockers should not be expected to interfere with their activity because this isoform of calcium channel is completely different to the cardiac and vascular versions.  Specifically, this version of the channel is directly mechanically coupled to the intracellular ryanodine receptor (RyR1), a calcium channel on the surface of the sarcoplasmic reticulum the mutant version of which is responsible for malignant hyperthermia, and which is the target of dantrolene. Because of the direct coupling between these two proteins, these voltage-gated calcium channels do not even need to conduct any calcium, and so most authors seem to refer to them as "dihydropyridine receptors" rather than channels in recognition of this fact. When an action potential activates them, their linked ryanodine receptors are also activated, hosing a stream of intracellular calcium directly at the nearby contractile apparatus (these structures all being conveniently positioned next to each other in a "triad"). The best reference for a detailed overview of this is Berchtold et al (2000).
• Cardiac myocytes, as mentioned above, have their own isoform of L-type voltage-gated calcium channels on the surface membrane of their T tubules, and these are also activated by the arrival of the action potential. These channels are not responsive to dihydropyridine, but verapamil and diltiazem can interfere in their activity, which forms the basis of their negative inotropic effect. These voltage-gated channels actually do conduct some calcium, in contrast to their counterparts in the skeletal muscle. Their opening allows the calcium concentration to increase locally at the dyadic space, the region of cytosol immediately below the surface of the tubule and above the membrane of the adjacent terminal cistern (those structures forming the dyad). Here, the calcium immediately acts on another type of ryanodine receptors (RyR2), which are calcium-gated calcium channels on the surface of the sarcoplasmic reticulum. The resulting amplification of intracellular calcium release is usually referred to as "calcium-induced calcium release" (CICR), and is the main mechanism by which cardiac myocytes get their calcium. This is the main difference between the excitation-contraction coupling of cardiac and skeletal muscle, explored in more detail by Eisner et al (2017).
• Smooth muscle have a totally different third variant of L-type voltage-gated calcium channels on the surface membrane, which get blocked by all kinds of calcium channel blocker drugs (wherein lies the origin of their antihypertensive effect). These also send some calcium into the cell in response to the arrival of an action potential, but smooth muscle cells are not uniformly dependent on this mechanism, and have multiple different ways of getting enough intracellular calcium, lovingly detailed by Berridge (2008). For example hormonal signals can allow calcium into the cell through membrane surface channels, or they can stimulate calcium release from endoplasmic reticulum stores, or the reticulum can release calcium without any external provocation whatsoever, by the rhythm of some kind of endogenous "calcium oscillator" effect that gets its periodicity modulated by hormonal controls. 

If you had to abbreviate the whole topic of excitation-contraction coupling to the description of just one mechanism, this would probably be it. These different methods of getting calcium into the myocyte are the main step that connects excitation to contraction, and if time were short, the exam candidate would probably be able to safely compress their answer around this central point and still score a few marks.

Role of calcium in the contractile apparatus 

Calcium is the final common pathway for all three muscle tissue types. It acts as a signalling molecule by interacting with regulatory proteins that modulate the activity of contractile proteins. Specifically, these regulatory proteins inhibit contraction, unless calcium comes along and turns them off. This mechanism is slightly different in skeletal cardiac and smooth muscle, and the clearest explanation of the differences actually comes from this old textbook chapter, around pages 237-238.

• Skeletal muscle uses troponin C as the regulator. Troponin C, upon binding calcium, undergoes a conformational change that pulls troponin I away from the actin/tropomyosin complex which permits these molecules to move. 
• Cardiac muscle also uses troponin C as the regulator,  except cardiac troponin C has a single low-affinity calcium binding site, whereas the skeletal muscle version has two.
• Smooth muscle uses calmodulin as the regulator. Calmodulin binds four calcium atoms, and activates myosin light chain kinase which then phosphorylates the light chain of myosin, greatly increasing its ATPase activity. The active myosin can then interact with actin and contract; whereas before phosphorylation it is in an inactive state.  This is a contrast to skeletal and cardiac muscle, where the ATPase activity of myosin is already constantly high, i.e. all skeletal and cardiac myosin is just waiting to be let off the leash by troponin C. Apart from calmodulin, smooth muscle also has other regulatory molecules (caldesmon, calponin) all of which also inhibit myosin ATPase activity and all of which are also deactivated in the presence of calcium.

Like in all cells, the calcium concentration in myocytes is closely regulated and the normal concentration of calcium in the cytosol is less than 200 nmol/L (Barry & Bridge, 1993). This is expected behaviour for an intracellular second messenger. Its concentration in the extracellular fluid (around 1.10-1.20 mmol/L) and in the sarcoplasmic reticulum (0.4 mmol/L) drives the gradient for its entry into the myocyte cytosol when the appropriate cation channels open. Though various authors (this one included) might occasionally refer to the resulting calcium influx as some kind of massive inundation, the reality is very different, as the cytoplasmic free calcium concentration might only increase up to something like 20 μmol/L, i.e. 0.02 mmol/L (Baylor & Hollingworth, 2003). This tiny fractional change is all that is required to produce muscle contraction.

ATP-dependent crossbridge cycling 

So: as mentioned above, with the regulatory brakes removed by calcium, contractile proteins are free to use as much ATP as they like in the process of contraction. That process is so well-described by such a vast range of excellent resources that there is probably nothing left to do but post an animated gif:

This "walk along" process can be explained with words, or images, or video, but (at least in the author's own personal experience) it does not seem to matter how many times, or in how many ways,  this process is explained - it will inevitably be forgotten by the vast majority of people shortly after whatever exam they are doing. Ergo, the most important consideration in presenting this information is to make it into easily crammable point-form statements for short term storage and easy regurgitation:

• Myosin and actin are associated at rest, with the myosin head at a 45º angle. 
• Then myosin binds ATP.
• This causes it to undergo a conformational change and dissociate from the actin.
• The hydrolysis of the ATP then causes myosin to "cock" its head to a 90º angle. 
  •  The ADP and the inorganic phosphate remain attached to the myosin
• This moves the myosin head about 11 nm along the actin filament.
• The cocked myosin head then binds the next actin active site (this  is the "cross-bridge")
  • Phosphate is then released from the myosin.
• The release of phosphate triggers the "power stroke":  a conformational change in the myosin which retuns it to the "uncocked" 45º angle position. 
• Because the myosin head was bound to the actin, this pulls the actin filament about 11 nm along the myosin molecule.
  • ADP is then released from the myosin
• This completes the cycle and locks the molecules in their new position, until the myosin head binds another ATP molecule. 

And it absolutely would bind another ATP molecule if it could, because the affinity of the myosin head for ATP is very high. In fact at physiologically normal levels of intracellular ATP the myosin head is never short of ATP, and would just keep repeating this process forever. For this reason we need regulatory molecules to prevent endless contraction. For striated muscle this occurs at the point of cross-bridge cycling, which is prevented by tropomyosin, and in smooth muscle the regulation occurs at the level of ATP hydrolysis which cannot occur until the myosin head is turned into an ATPase by calmodulin. All of these regulatory mechanisms require the absence of calcium to function, which means calcium needs to be rapidly removed from the cytosol to allow relaxation. This is achieved by two main systems: calcium buffering and the sequestration of calcium in the sarcoplasmic reticulum.

Activation of the sarcoplasmic calcium buffering system

Calcium buffering is a method of rapidly decreasing the calcium concentration in the myocyte that requires basically no energy, which is very attractive. As a divalent cation calcium is already attracted to negatively charged regions on protein molecules (just look at how it interacts with albumin in the bloodstream), so to associate with some cytosolic proteins and become biologically inactive would not be a huge change in its normal behaviour. The most important buffers are troponin C itself, ATP, and parvalbumin - a protein that seems to be specifically purposed to buffer calcium, and which acts as a chaperone to deliver it to the sarcoplasmic reticulum. This mechanism plays a variable role in different muscle tissues; it is probably more valuable to fast-twitch skeletal muscle where rapid relaxation is necessary (as these seem to contain more parvalbumin). 

Calcium sequestration 

Ultimately, binding calcium to cytosolic proteins is not a long-term solution for the intracellular calcium problem. All those ions still need to be disposed of somehow to abolish contraction and to return the muscle to the relaxed state. There are two options: either they get deported by surface membrane pumps, or they get confined in the sarcoplasmic reticulum. The latter is the dominant mechanism, and both options are explored in considerable detail by Berchtold et al (2000).

Transmembrane pumps like the sodium-calcium exchange pump NCX or the ATP-powered calcium transporter PMCA do contribute, but conceptually it would be hard to justify moving all the calcium into the extracellular fluid because it is such an important second messenger, and it would not be beneficial to the organism to have huge exercise-associated fluctuations in calcium levels. It would be better to sequester it into the sarcoplasmic reticulum, and this movement is mediated by the ATP-powered SERCA pumps, where "SERCA" stands for "sarco(endo)plasmic reticulum calcium ATPase". This thing cracks enough ATP molecules to account for something like 40-50% of resting muscle metabolism, or 12-15% of total body oxygen consumption, considering that muscle mass represents perhaps 40% of total body mass. 

The function of this pump is regulated by various mechanisms, which differ between muscle tissue types, and only one of which has some vague pharmacological interest for the intensivist. In cardiac muscle, the protein phospholamban inhibits SERCA activity by reducing its affinity for calcium, thus preventing relaxation (MacLennan et al, 2003). However, when demand for cardiac output is increased, the myocardium would need to relax more, not less, to accommodate a larger end-diastolic volume. This is achieved by catecholamines, as they activate protein kinase A which phosphorylates phospholamban and causes it to dissociate form SERCA. The disinhibited SERCA then gobbles up all the calcium and you get a bigger stroke volume to go with your increased contractility. This is the mechanism of catecholamine-induced increase in lusitropy. 

Relaxation is therefore an ATP dependent process, as ATP-powered pumps are necessary to remove calcium and allow normal inhibitory activity of cross-bridge cycling regulators. Also the myosin heads need new ATP to dissociate from actin. 

In death, where all the myosin heads run out of ATP and there is nobody left to remove calcium from the cytosol, the muscle proteins end up stuck in a rigid contracted state. This is the basis for rigor mortis, which develops early after death, and which persists for perhaps 15-25 hours (until the contractile proteins start to get degraded by lysosomal enzymes).

Relaxation in smooth muscle

Merely removing calcium from the cytosol may not stop the process of smooth muscle contraction by itself, as the phosphorylated myosin will just carry on with its ATPase activity even in the absence of calcium. It needs to be dephosphorylated in order for it to stop. This is achieved by myosin light chain phosphatase, an enzyme that is an indirect target for smooth muscle relaxants that act via the nitric oxide and cGMP pathways. Nitric oxide, a soluble extracellular mediator, penetrates into cells and activates guanylate cyclase, which produces cyclic GMP, which then activates protein kinase G, which in turn phosphorylates myosin light chain phosphatase, activating it and bringing about smooth muscle relaxation.